1. Field of the Invention
The invention relates to a method of obtaining endomycorrhizian fungi with vesicles and arbuscules (VAM) in vitro. More specifically, the present invention relates to the inoculation of genetically converted dicotyledon roots with endomycorrhiza spores.
2. Description of the Prior Art
Recent developments in research pertaining to endotrophic mycorrhizas (endomycorrhizas) have shown their use in agriculture and horticulture to have great potential importance. These endophytic fungi are far from the most common, but come into symbiosis with virtually all cultivated plants. They are also characterized by at least three basic properties:
(i) their association with a plant is essential to their own development, PA0 (ii) they have a wide range of host plants, and no specificity has so far been recorded, and, PA0 (iii) they stimulate growth of the plants which they infect.
The favorable effect on the host plant is a well established fact. It is due to an increase in absorption of inorganic ions by the mycorrhized plant, and more particularly, the absorption of relatively immobile ions and chiefly phosphorus ones. It has been demonstrated that the hyphas of the endomycorrhizogenic fungus are capable of absorbing soluble phosphorus, transporting it and transferring it to the plant. These processes lead to improved growth of the mycorrhized plant. See, for example, GIANINAZZI S., 1976 Physiol. Veg., Vol. 14, 733-741.
Furthermore, it has been observed that infection by an endomycorrhizian fungus may protect the roots of the mycorrhized plant from pathogens in the soil. For example, see, BALTRUSCHAT H., 1975, Phytopathol, Vol. 84, 172-188.
However, despite the large amount of work carried out to date, e.g., RHODES L. H., 1980, Outlook Agricult., Vol. 10, 275-281; HAYMAN D. S., 1980 Nature, Vol. 287, 487-490, the main difficulty in applying endotrophic mycorrhizas is that it is not known how to produce them in large quantities for commercial application. It is believed that no one has ever succeeded in cultivating them in a sterile medium without the host plant.
Unlike most symbiotic micro-organisms which are parasitic or saprophytic, VAM fungi cannot be cultivated in vitro. Note, BARRET J. T., 1947, Phytopathol., Vol. 37, 359; MOOSE B., 1962, J. Gen. Microbiol., Vol. 27, 509-520; HARLEY J. L., 1965, Ecology of Soil-Borne Plant Pathogens, Baker and Snyder Eds., Univ. California Press, 218-229; HEPPER C. M., 1979, Soil Biol. Biochem., Vol. 11, 269-277; HEPPER C. M., 1981, New Phytol., Vol. 88, 641-647. The large number of attempts at mycorrhization which have been carried out so far have consisted of using inoculums prepared from complete plants which are cultivated in pots or a greenhouse. Inoculation is nearly always carried out with specially gathered mycorrhized roots, or sometimes with a suspension of spores. JACKSON et al, 1972, Proc. Soil Sci. Soc. Am., Vol. 36, 64-67, used lyophilised roots, while HALL, 1979, Soil Biol. Biochem., Vol. 11, 85-86, recommends using soil pellets mixed with infected roots.
In all cases, one of the problems encountered is the possibility of introducing contaminants into the soil through inoculation of the VAM, particularly when for reasons of plant health it is necessary to disinfect the soil before planting, thereby resulting in destruction of the VAM naturally present. Moreover, the time required to produce an inoculum is generally two-four months, which is long enough to produce many contaminants. Such problems have hindered commercialization of the inoculums. In addition, the inoculums cost at least as much as, or more than, the fertilizers which would be required to obtain the same yield. Consequently the large scale use of endomycorrhizas has not yet been adopted in agricultural practice due to the problems of producing an inoculum which is plentiful, free of contamination, yet easy to store and easy to handle on the land.
In an attempt to resolve this problem, various studies have been carried out on the behavior in vitro of VAM fungi, associated with complete plants or roots. Although these methods are very helpful in studying the metabolism of VAM, they do appear to be uncertain as a means of producing fungus in pure culture, owing to the very slow development of the roots. Note, for example, HEPPER C. M., 1975, in Endomycorrhizas, Sander F. E., Mosse B. and Tinker, P. E. Eds, Acad. Press London, 67-86; MOSSE B., and HEPPER C. M., 1975, Physiol. Pl. Pathol., Vol. 5, 215-225; HEPPER C. M., 1981, New Phytol., Vol. 88, 641-647; MACDONALD R. M., 1981, New Phytol., Vol. 89, 87-93; ST. JOHN T. V., and REID, C. P. P., 1981, New Phytol., Vol. 89, 81-86.
It is known that genetic conversion of roots by insertion of a fragment of foreign DNA (known as T-DNA) from the root inducing plasmid of Agrobacterium rhizogenes enables aseptic cultures of roots to be formed with rapid and indefinite growth in a simple medium. Compare TEPFER D. A., 1981, C.R. Acad. Sc. PARIS, t. 292. Serie III, 153. Agrobacterium rhizogenes is the agent of a disease, i.e., hairy-root, the symptom of which is proliferation of a large number of root hairs at the point where the bacterium is inoculated.
Accordingly, it is an object of the present invention to overcome the problems of producing endomycorrhizas on a commercial scale by providing a successful method for production thereof.
It is another object of the present invention to overcome the aforenoted problems by using the new powers conferred to roots converted by the action of Agrobacterium rhizogenes, through inoculating cultures of such converted roots with spores of endomycorrhizas.
In particular, an object of the present invention is to provide a method of obtaining endomycorrhizas in vitro, particularly in a fermenter.
Other objects of the present invention will become readily apparent upon a review of the following description, the figures of the drawing and the claims appended hereto.